Compositions corresponding to a calcium transporter and methods of making and using same

ABSTRACT

Nucleic-acid and amino-acid sequences correspond to a calcium-transport protein regulating the movement of calcium across cell membranes.

REFERENCE TO RELATED APPLICATIONS

[0001] This is a divisional of prior application Ser. No. 09/350,457,filed Jul. 9, 1999, the entire disclosure of which is incorporated byreference herein.

FIELD OF THE INVENTION

[0002] The present invention relates to transcellular transport ofcalcium, and in particular to compositions encoding calcium-transportproteins.

BACKGROUND OF THE INVENTION

[0003] Calcium is a major component of the mineral phase of bone, and inionic form plays an important role in cellular signal transduction. Inparticular, a signaling ligand (the “first messenger”) such as a hormonemay exert an effect on a cell to which it binds by causing a short-livedincrease or decrease in the intracellular concentration of anothermolecule (the “second messenger”); calcium is known to play the role offirst or second messenger in numerous cellular signaling contexts.

[0004] Calcium homeostasis in blood and other extracellular fluids istightly controlled through the actions of calciotropic hormones on bone,kidneys, and intestine. In humans, dietary intake of calciumapproximates 500 to 1000 mg/day, and obligatory endogenous losses instool and urine total about 250 mg/day. On the order of 30% of calciumin the diet must be absorbed to sustain bone growth in children and toprevent postmenopausal bone loss in aging women. To meet the body's needfor calcium, the intestines of most vertebrates evolved specializedvitamin D-dependent and -independent mechanisms for ensuring adequateintestinal calcium uptake. Intestinal absorption of Ca²⁺ occurs by botha saturable, transcellular process and a nonsaturable, paracellularpathway. When dietary calcium is abundant, the passive paracellularpathway is thought to be predominant. In contrast, when dietary calciumis limited, the active, vitamin D-dependent transcellular pathway playsa major role in calcium absorption.

[0005] The transcellular intestinal-uptake pathway is a multistepprocess, consisting of entry of luminal Ca²⁺ into an intestinalepithelial cell (i.e., an enterocyte), translocation of Ca²⁺ from itspoint of entry (the microvillus border of the apical plasma membrane) tothe basolateral membrane, followed by active extrusion from the cell.Intracellular Ca²⁺ diffusion is thought to be facilitated by a calciumbinding protein, calbindin D_(9K), whose biosynthesis is dependent onvitamin D. The extrusion of Ca²⁺ takes place against an electrochemicalgradient and is mainly mediated by Ca-ATPase. The entry of Ca²⁺ acrossthe apical membrane of the enterocyte is strongly favoredelectrochemically because the concentration of Ca²⁺ within the cell(10⁻⁷-10⁻⁶ M) is considerably lower than that in the intestinal lumen(10⁻³ M) and the cell is electronegative relative to the intestinallumen; as a result, the movement of Ca²⁺ across the apical membrane doesnot require the expenditure of energy.

[0006] The molecular mechanism responsible for entry of Ca²⁺ intointestinal cells has, however, been difficult to characterize. Inparticular, researchers have disagreed as to whether a transporter or achannel is responsible for this process (although studies have indicatedthat Ca²⁺ entry is voltage-independent and largely insensitive toclassic L-type calcium channel blockers).

DESCRIPTION OF THE INVENTION BRIEF SUMMARY OF THE INVENTION

[0007] The present invention is directed, in a first aspect, toward amembrane protein that functions to transport calcium across cellularmembranes. Our data indicate that this protein plays a key role inmediating Ca²⁺ entry into enterocytes as the first step of transcellularintestinal calcium absorption. Expression of the human homologue can bedetected in placenta, pancreas, prostate, kidney, the gastrointestinaltract (e.g., esophagus, stomach, duodenum, jejunum, colon), liver, hairfollicles, and testis, and is also expressed in cancer cell lines(specifically, chronic myelogenous leukemia cell line K-562 andcolorectal adenocarcinoma cell line SW480). The rat isoform is expressedin rat intestine (although not in rat kidney). Thus, in contrast to therat isoform, the human protein may be involved in theabsorption/resorption of calcium in both intestine and kidney.Dysfunction of the human protein may be implicated in hyper- andhypocalcemia and calciuria, as well as in bone diseases, leukemia, andcancers affecting the prostate, breast, esophagus, stomach, and colon.

[0008] One embodiment of the invention comprises, as a composition ofmatter, a non-naturally occurring calcium-transport protein. Preferably,the transporter is a polypeptide encoded by a nucleic acid sequencewithin Seq. I.D. No. 1 or 3. In this context, the term “encoded” refersto an amino-acid sequence whose order is derived from the sequence ofthe nucleic acid or its complement. The nucleic acid sequencerepresented by Seq. I.D. No. 1 is derived from human sources. Thenucleic acid sequence represented by Seq. I.D. No. 3 is derived fromrat.

[0009] One aspect of this embodiment is directed toward a transporterhaving an amino-acid sequence substantially corresponding at least tothe conserved regions of Seq. I.D. Nos. 2 or 4. The term“substantially,” in this context, refers to a polypeptide that maycomprise substitutions and modifications that do not alter thephysiological activity of the protein to transport calcium acrosscellular membranes. The polypeptide represented by Seq. I.D. No. 2 isderived from human sources. The peptide represented by Seq. I.D. No. 4is derived from rat.

[0010] In a second aspect, the invention pertains to a non-naturallyoccurring nucleic acid sequence encoding a calcium-transport protein.One embodiment of this aspect of the invention is directed toward atransporter having a nucleotide sequence substantially corresponding atleast to the conserved regions of Seq. I.D. Nos. 2 or 4. The term“substantially,” in this context, refers to a nucleic acid that maycomprise substitutions and modifications that do not alter encoding ofthe amino-acid sequence, or which encodes a polypeptide having the samephysiological activity in transporting calcium across cellularmembranes. The term “corresponding” means homologous or complementary toa particular nucleic-acid sequence.

[0011] As used herein, the term “non-naturally occurring,” in referenceto a cell, refers to a cell that has a non-naturally occurring nucleicacid or a non-naturally occurring polypeptide, or is fused to a cell towhich it is not fused in nature. The term “non-naturally occurringnucleic acid” refers to a portion of genomic nucleic acid, a nucleicacid derived (e.g., by transcription) thereof, cDNA, or a synthetic orsemi-synthetic nucleic acid which, by virtue of its origin or isolationor manipulation or purity, is not present in nature, or is linked toanother nucleic acid or other chemical agent other than that to which itis linked in nature. The term “non-naturally occurring polypeptide” or“non-naturally occurring protein” refers to a polypeptide which, byvirtue of its amino-acid sequence or isolation or origin (e.g.,synthetic or semi-synthetic) or manipulation or purity, is not presentin nature, or is a portion of a larger naturally occurring polypeptide,or is linked to peptides, functional groups or chemical agents otherthan that to which it is linked in nature.

[0012] A third aspect of the present invention comprises a method oftransporting calcium across a cellular membrane having a calciumtransporter in accordance herewith. Calcium (in the divalent ionic form)is applied to the cellular membrane under conditions that allow thetransporter to transport the calcium.

[0013] The cellular membrane can be derived, for example, from placenta,pancreas, prostate, kidney, the gastrointestinal tract (e.g., esophagus,stomach, duodenum, jejunum, colon), liver, or testis; or may be one ofthese tissues either in vivo or ex vivo. In practicing the method, thecell(s) giving rise to the cellular membrane may be transformed with thenucleic acid of Seq. I.D. Nos. 1 or 3 and maintained under conditionsfavoring functional expression of the transporter. A cell may bemonitored for expression of the transporter by measuring the presence ofcalcium in the cell or transmembrane current flow. The invention alsoextends to a cell so transformed (e.g., a Xenopus laevis oocyte asdescribed below).

[0014] In a fourth aspect, the invention comprises a method ofidentifying chemicals capable of interacting with the transporter,whether the protein is integral with a cellular membrane or present as afree species. Such chemicals may include antibodies or other targetingmolecules that bind to the protein for purposes of identification, orwhich affect (e.g., by modulation or inhibition) the transportproperties of the protein; and transportable species other than calcium.

[0015] In a fifth aspect, the invention comprises a method of blockingor inhibiting the uptake of calcium by cells having a calcium-transportprotein in accordance herewith. In one embodiment, the method comprisesthe steps of causing an antibody or other targeting molecule to bind tothe protein in a manner that inhibits calcium transport. In anotherembodiment, a nucleic acid complementary to at least a portion of thenucleic acid encoding the calcium-transport protein is introduced intothe cells. The complementary nucleic acid blocks functional expressionof the calcium-transport protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The foregoing discussion will be understood more readily from thefollowing detailed description of the invention, when taken inconjunction with the accompanying drawings, in which:

[0017]FIG. 1 graphically illustrates identification of CaT1 in Xenopuslaevis oocytes by means of a calcium-uptake assay;

[0018]FIGS. 2A and 2B schematically illustrate the structure andtopology of a human and a rat calcium transporter, respectively, inaccordance herewith;

[0019]FIGS. 3A and 3B illustrate the primary peptide structure of ahuman calcium transporter in accordance herewith;

[0020]FIGS. 4A and 4B illustrate the primary peptide structure of a ratcalcium transporter in accordance herewith;

[0021] FIGS. 5A-5E graphically illustrate various calcium-uptakeproperties of rat CaT1 proteins;

[0022]FIGS. 6A and 6B depict responses of Xenopus laevis oocytesexpressing CaT1 following external application of Ca²⁺;

[0023]FIGS. 6C and 6D depict responses of Xenopus laevis oocytesexpressing CaT1 following injection of the calcium chelator EGTA (i.e.,ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid);

[0024]FIG. 7A depicts the response of Xenopus laevis oocytes expressingCaT1 to Na⁺ in the absence of Ca²⁺;

[0025]FIG. 7B depicts the response of Xenopus laevis oocytes expressingCaT1 to Ca²⁺ in the presence of Na⁺ at low concentrations;

[0026]FIGS. 8A and 8B depict the charge-to-⁴⁵ Ca⁺ ratio involtage-clamped, CaT1-expressing oocytes in the presence of and in theabsence of Na⁺, respectively; and

[0027]FIG. 9 depicts the response of voltage-clamped, CaT1-expressingoocytes to Ca²⁺, Ba²⁺, Sr²⁺ and Mg²⁺.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] 1. CaT1 and its Nucleic Acids

[0029] With reference to Seq. I.D. No. 1, a human-derived cDNA having anucleotide sequence encoding a calcium-transport protein contains 2218nucleotides; an open reading frame of 2175 base pairs (bp) encodes aprotein having 725 amino acids, which is set forth as Seq. ID. No. 2.With reference to Seq. I.D. No. 3, a rat-derived cDNA having anucleotide sequence encoding a calcium-transport protein contains 2995nucleotides; an open reading frame of 2181 base pairs (bp) encodes aprotein having 727 amino acids, which is set forth as Seq. ID. No. 4.This protein is set forth as Seq. ID. No. 4. The designation CaT1 isherein used interchangeably to refer to the human protein of Seq. I.D.No. 2 and the rat protein of Seq. I.D. No. 4.

[0030] The predicted relative molecular mass of rat CaT1 is 83,245(M_(r)=83.2 kDa), which is consistent with the molecular weight obtainedby in vitro translation without microsomes (84 kDa). Hydropathy analysissuggests that the calcium transporter is a polytopic protein containingsix transmembrane domains (TMs) with an additional short hydrophobicstretch between TM5 and 6 as illustrated in FIG. 1. Consistent with themolecular weight of the protein obtained by in vitro translation in thepresence of microsomes (89 kDa), an N-glycosylation site is predicted inthe first extracellular loop of the protein. The amino-terminalhydrophilic segment (326 amino acid residues) of rat CaT1 contains fourankyrin repeat domains, suggesting that the protein may somehowassociate with the spectrin-based membrane cytoskeleton. The carboxylterminus (150 amino-acid residues) contains no recognizable motifs.Putative phosphorylation sites for protein kinases A (PKA) and C (PKC)are present in the cytoplasmic domains, suggesting that transportactivity could be regulated by phosphorylation. FIGS. 2 and 3 illustratethe primary structures of human and rat CaT1, respectively, showing thetransmembrane domains as well as glycosylation, PKA, and PKC sites, andankyrin repeat sequences.

[0031] The human protein shows 75% amino acid sequence identity to therecently cloned rabbit apical epithelial calcium channel ECaC (seeHoenderop et al., J. Biol. Chem. 296:8375-8378 (1999)) when using theBESTFIT sequence alignment program. There are, however, numerousdifferences between the proteins, in particular with respect to theamino- and carboxyl-terminal cytoplasmic domains, which are considerablymore conserved between the rat and human CaT1 than between eithertransporter and ECaC; the number of ankyrin repeats; the number anddistribution of PKA and PKC phosphorylation sites; and theirN-glycosylation sites. In particular, the amino- and carboxyl-terminalcytoplasmic domains of CaT1 from rat and ECaC from rabbit exhibit alower degree of similarity than the equivalent regions of rat CaT1 andpartial sequences obtained from human small intestine (not shown) byhomology screening using the CaT1 cDNA as a probe. Comparisons ofsequences of 150 amino acids in the amino- or carboxyl-terminalcytoplasmic domains revealed 90% and 74% identities respectively,between rat and human CaT1 but only 61% and 50% identities,respectively, between CaT1 and ECaC. CaT1 has four ankyrin repeats andone PKA phosphorylation site in its amino-terminal segment, whereas ECaCcontains three ankyrin repeats and no PKA site in the same region. Incontrast, ECaC possesses three PKC sites and two PKA sites in itscarboxyl-terminus, whereas CaT1 has only one PKC site and no PKA sitesin the same region. In addition, CaT1 lacks the putative N-glycosylationsite found in ECaC between the pore region and transmembrane domain 6. Astriking difference between CaT1 and ECaC is that ECaC is abundant inthe distal tubules and cortical collecting duct of rabbit kidney, whilethe CaT1 mRNA was undetectable in rat kidney, based on Northern analysisand in situ hybridization.

[0032] Additional homology searches of available protein databasesrevealed significant similarities between CaT1 and the capsaicinreceptor, VR1 (see Caterina et al., Nature 389:816-824 (1997)), andOSM-9, a C. elegans membrane protein involved in olfaction,mechanosensation and olfactory adaptation (see Colbert et al., J.Neurosci. 17:8259-8269 (1997)). These proteins are structurally relatedto the family of putative store-operated calcium channels, among whichthe first two identified were the Drosophila retinal proteins, TRP (seeMontell et al., Neuron 2:1313-1323 (1989)) and TRPL (see Phillips etal., Neuron 8:631-642 (1992)). Based on the program BESTFIT, CaT1 shows33.7% and 26.7% identities to VR1 and OSM-9, respectively, over astretch of at least 500 residues, as well as 26.2% and 28.9% identitiesto TRP and TRPL, respectively, in more restricted regions (residues552-593 for TRP and residues 556-593 for TRPL). The latter region coverspart of the pore region and the last transmembrane domain. A commonfeature of all of these proteins is the presence of six TMs with ahydrophobic stretch between TM5 and TM6, resembling one of the fourrepeated motifs of 6 TMs in the voltage-gated channels. Another commonfeature is the presence of three to four ankyrin repeat domains in thecytoplasmic N-terminal region. Of note, members of the polycystin familyalso possess 6 transmembrane segments (30-32) and show a modest degreeof homology to CaT1 in small regions of the predicted amino acidsequences (residues 596-687 in PKD2, 23% identity; and residues 381-483in PKD2L, 26% identity), but the polycystins contain no ankyrin repeats.As explained below, however, it is unlikely that CaT1 is another subtypeof capsaicin-gated or store-operated ion channels.

[0033] A homology search using the CaT1 sequence in expressed sequencetag (EST) databases revealed the following sequences with high degreesof similarity to CaT1 (names refer to GenBank accession numbers and %identities to nucleotide identities): AI101583 from rat brain (99%);AI007094 from mouse thymus (96%); AA447311, AA469437, AA579526 fromhuman prostate (87%, 85%, 84%, respectively), W88570 from human fetalliver spleen (91%); AA078617 from human brain (85%); T92755 from humanlung (92%).

[0034] 2. Isolation and Analysis of CaT1

[0035] To clone the gene(s) encoding CaT1, an expression cloningstrategy using Xenopus laevis oocytes as the expression system wasemployed. Functional screening of a rat duodenal library by measuring⁴⁵Ca²⁺ uptake resulted in the isolation of a cDNA clone encoding CaT1.We found that oocytes injected with mRNA from rat duodenum or cecumexhibited reproducible increases in Ca²⁺ uptake over water-injectedcontrol oocytes. After size-fractionation of rat duodenal poly(A)⁺ RNA,we detected a substantial increase in ⁴⁵Ca²⁺ uptake by injection of RNAfrom a 2.5 to 3 kb pool (FIG. 1). A library was constructed using thisRNA pool, and a single clone was isolated from this size-fractionatedcDNA library by screening progressively smaller pools of clones fortheir ability to induce ⁴⁵Ca²⁺ uptake in cRNA-injected oocytes. Theresultant 3-kb cDNA produced large increases in Ca²⁺ uptake (˜30 fold)when expressed in oocytes.

[0036] The experimental procedures we employed were as follows.

[0037] Expression cloning—Expression cloning using Xenopus oocytes wasperformed in accordance with known techniques as described in Romero etal., Methods Enzymol. 296:17-52 (1998), hereby incorporated byreference. In particular, duodenal poly(A)⁺ RNA from rats fed acalcium-deficient diet for 2 weeks was size-fractionated. A cDNA librarywas then constructed from the fractions of 2.5 to 3 kilobases (kb) thatstimulated ⁴⁵Ca²⁺ uptake activity when expressed in oocytes. The RNAssynthesized in vitro from pools of ˜500 clones were injected intooocytes, and the abilities of the pools to stimulate Ca²⁺ uptake wereassayed. A positive pool was sequentially subdivided and assayed in thesame manner until a single clone was obtained. The cDNA clone wassequenced bidirectionally.

[0038]⁴⁵Ca²⁺ uptake assay—Defolliculated Xenopus laevis oocytes wereinjected with either 50 nl of water or RNA. ⁴⁵Ca²⁺ uptake was assayed 3days after injection of poly(A)⁺ or 1-3 days after injection ofsynthetic complementary RNA (cRNA). For expression cloning, oocytes wereincubated in modified Barth's solution supplemented with 1 mM SrCl₂ (toavoid excessive loading of oocytes with Ca²⁺) as well as penicillin,streptomycin and gentamycin at 1 mg/ml. Standard uptake solutioncontained the following components (in mM): NaCl 100, KCl 2, MgCl₂ 1,CaCl₂ 1 (including ⁴⁵Ca²⁺), Hepes 10, pH 7.5. Uptake was performed atroom temperature for 30 minutes (for the expression cloning procedure, 2hour uptakes were employed), and oocytes were washed 6 times withice-cold uptake medium plus 20 mM MgCl₂. The effects of capsaicin orL-type channel blockers on Ca²⁺ uptake were studied in uptake solutionby addition of 50 μM capsaicin (in ethanol solution, final concentration0.05%) or 10-100 μM calcium channel blockers in water (nifedipine wasdiluted with uptake solution from 100 mM DMSO stock solution). Controlexperiments were performed with the appropriate ethanol and DMSOconcentrations. Unless stated specifically, data are presented as meansobtained from at least three experiments with 7 to 10 oocytes per groupwith standard error of the mean (S.E.M.) as the index of dispersion.Statistical significance was defined as having a P value of less than0.05 as determined by Student's t-test.

[0039] In situ hybridization—Digoxigenin-labeled sense and antisenserun-off transcripts were synthesized. CaT1 cRNA probes were transcribedfrom a PCR fragment that contains about 2.7 kb of CaT1 cDNA (nucleotides126-2894) flanked at either end by promoter sequences for SP6 and T7 RNApolymerases. Sense and anti-sense transcripts were alkali-hydrolyzed toan average length of 200-400 nucleotides. In situ hybridization wasperformed on 10-μm cryosections of fresh-frozen rat tissues. Sectionswere immersed in slide mailers in hybridization solution composed of 50%formamide, 5×SSC, 2% blocking reagent, 0.02% SDS and 0.1%N-laurylsarcosine, and hybridized at 68° C. for 16 hours with sense orantisense probe at a concentration of about 200 ng/ml. Sections werethen washed 3 times in 2×SSC and twice for 30 min in 0.2×SSC at 68° C.After washing, the hybridized probes were visualized by alkalinephosphatase histochemistry using alkaline-phosphatase-conjugatedanti-digoxigenin Fab fragments and bromochloroindolylphosphate/nitroblue tetrazolium (BCIP/NBT).

[0040] In vitro transcription was performed with the mMESSAGE mMACHINET7 Kit (Ambion, Austin, Tex.). In vitro translation of the CaT1 proteinwas performed with the Rabbit Reticulocyte Lysate System (Promega,Madison, Wis.).

[0041] 3. Tissue Distribution of CaT1

[0042] Northern analysis of rat tissues revealed a strong 3.0-kb band inrat small intestine and a weaker 6.5-kb band in brain, thymus andadrenal gland. No CaT1 transcripts were detected in heart, kidney,liver, lung, spleen and skeletal muscle. Northern analysis of thegastrointestinal tract revealed that the 3-kb CaT1 transcript isexpressed in duodenum and proximal jejunum, cecum and colon but not instomach, distal jejunum or ileum. The CaT1 mRNA in rat duodenum was notregulated by 1,25-dihydroxyvitamin D₃ nor by calcium deficiency in vivo.

[0043] In situ hybridization revealed expression of CaT1 mRNA in theabsorptive epithelial cells of duodenum, proximal jejunum, cecum andcolon but not in ileum. CaT1 mRNA is expressed at high levels induodenum and cecum, at lower levels in proximal jejunum and at very lowlevels in colon. In all CaT1-expressing intestinal segments, mRNA levelswere observed to be higher at the villi tips than in the villi crypts.No signals were detected in the kidney under the same experimentalconditions or in sense controls.

[0044] Northern analysis procedures were as follows. Poly(A)⁺ RNA (3 μg)from rat tissues were electrophoresed in formaldehyde-agarose gels andtransferred to nitrocellulose membranes. The filters were probed with³²P-labeled full-length CaT1 cDNA, hybridized at 42° C. with a solutioncontaining 50% formamide, 5×SSPE, 2× Denhardt's solution, 0.1% SDS and100 μg/ml denatured salmon sperm DNA (and washed with 5×SSC/0.1% SDS at50° C. for 2×30 minutes and 0.1×SSC at 65° C. for 3×30 minutes.Autoradiography was performed at −80° C. for 1 to 2 days.

[0045] 4. Characterization of Functional Properties of CaT1 by ⁴⁵Ca²⁺Uptake Assay

[0046] Since CaT1 shares some similarity in its structure with thecapsaicin receptor (VR1), TRP and TRPL channels, we tested thepossibility that the activity of CaT1 could be stimulated by capsaicinor calcium-store depletion using the uptake assay described above.Capsaicin (up to 50 μM) did not stimulate CaT1-mediated ⁴⁵Ca²⁺ uptake inoocytes. Instead of stimulating Ca²⁺ entry, depletion of calcium storesby thapsigargin treatment decreased CaT1-mediated Ca²⁺ activity to about20% of its baseline activity (FIG. 5A). Based on these data, it isunlikely that CaT1 is another subtype of capsaicin-gated orstore-operated ion channels.

[0047] When expressed in oocytes, CaT1-mediated ⁴⁵Ca²⁺ uptake was linearfor up to 2 hours. Ca²⁺ uptake was concentration-dependent andsaturable, with an apparent Michaelis constant (K_(m)) of 0.44±0.07 mM(FIG. 5B). This K_(m) is appropriate for absorbing Ca²⁺ from theintestine, which is normally around 1 to 5 mM after a calcium-containingmeal, and accords with values reported in physiological studies ofcalcium absorption in rat, hamster, pig, and human intestines.Consistent with the prediction from early studies that apical Ca²⁺uptake is not energy-dependent, CaT1-mediated transport did not appearto be coupled to Na⁺, Cl⁻ or H⁺ (FIGS. 5C and 5D). To study thesubstrate specificity of CaT1, we initially performed inhibition studiesof ⁴⁵Ca² uptake (1 mM Ca²⁺) by various di- and trivalent cations (100μM) (FIG. 5E). Gd³⁺, La³⁺, Cu²⁺, Pb²⁺, Cd²⁺, Co²⁺ and Ni²⁺ producedmarked to moderate inhibition, whereas Fe²⁺, Fe³⁺, Mn²⁺ and Ni²⁺ had nosignificant effects. In contrast, Ba²⁺ and Sr²⁺ had only slightinhibitory effects, even at a concentration of 10 mM, whereas Mg²⁺ (10mM) produced no significant inhibition (FIG. 5E).

[0048] Ca²⁺ entry into enterocytes has, in general, been reported to beinsensitive to classic voltage-dependent calcium channel blockers, andto be only slightly inhibited by verapamil. Among the three classes ofL-type calcium channel blockers that we tested—nifedipine, diltiazem andverapamil—only the latter two modestly inhibited CaT1-mediated Ca²⁺uptake (by 10-15%) at relatively high concentrations (10-100 μM).

[0049] 5. Electrophysiological Properties of CaT1-Mediated transport

[0050] Two-microelectrode voltage clamp experiments were performed usingstandard techniques (see Chen et al., J. Biol. Chem. 274:2773-2779(1999)) using a commercial amplifier and pCLAMP software (Version 7,Axon Instruments, Inc., Foster City, Calif.). An oocyte was introducedinto the chamber containing Ca²⁺-free solution and was incubated forabout 3 minutes before being clamped at −50 mV and subjected tomeasurements. In experiments involving voltage ramps or jumps,whole-cell current and voltage were recorded by digitizing at 300μs/sample and by Bessel filtering at 10 kHz. When recording currents ata holding potential, digitization at 0.2 s/sample and filtering at 20 Hzwere employed. Voltage ramping consisted of pre-holding at −150 mV for200 ms to eliminate capacitive currents and a subsequent linear increasefrom −150 to +50 mV, with a total duration of 1.4 s. Voltage jumpingconsisted of 150 ms voltage pulses of between −140 and +60 mV, inincrements of +20 mV. Steady-state currents were obtained as the averagevalues in the interval from 135 to 145 ms after the initiation of thevoltage pulses. For experiments involving voltage-clamped ⁴⁵Ca²⁺ uptake,Ca²⁺-evoked currents and uptake of ⁴⁵Ca⁺ were simultaneously measured at−50 mV, using a method similar to that described in Chen et al. (citedabove).

[0051] It is found that CaT1-mediated Ca²⁺ transport is driven by theelectrochemical gradient of Ca²⁺. There is no evidence for coupling ofCa²⁺ uptake to other ions or to metabolic energy. While CaT1-mediatedCa²⁺ transport is electrogenic and voltage dependent, its kineticbehavior is distinct from that of the voltage-dependent calciumchannels, which are operated by membrane voltage. At a macroscopiclevel, the kinetic properties of CaT1 resemble those of a facilitatedtransporter, and patch clamp studies have not as yet provided anyevidence for distinct single-channel activity. CaT1 may represent anevolutionary transition between a channel and a facilitated transporter.

[0052] More specifically, external application of Ca²⁺ to oocytesexpressing CaT1 generated inward currents at a holding potential of −50mV (FIG. 6A), which were absent in control oocytes. Addition of 5 mMCa²⁺ evoked an overshoot of inward current to several hundred nAfollowed by a rapid reduction to a plateau value of 20-50 nA (FIG. 6A).CaT1-mediated current was also voltage-dependent, as revealed bycurrent-voltage (I-V) curves (FIG. 6B). The peak current is due toendogenous Ca²⁺-activated chloride-channel currents because it could beblocked by chloride channel blockers such as flufenamate. The plateaualso contained flufenamate-inhibitable currents, suggesting that someendogenous, Ca²⁺-activated chloride channels remained active during thisphase. Chelating intracellular Ca²⁺ by injection of EGTA into oocytesexpressing CaT1 to a final concentration of 1-2 mM resulted in a three-to five-fold increase in Ca²⁺ uptake and abolished the overshoot of thecurrent (FIG. 6A). Under the same condition, EGTA-injected controloocytes produced no detectable currents. Therefore, CaT1 likely mediatesthe observed Ca²⁺-evoked currents in EGTA-injected oocytes (FIGS. 6C,6D).

[0053] In the absence of Ca²⁺, oocytes expressing CaT1 exhibited asignificant permeability to Na⁺ at hyperpolarized potentials (FIG. 7A).Similar conductances were observed for K⁺, Rb+ and Li⁺(K⁺≈Rb⁺>Na⁺>Li⁺).CaT1-mediated permeation of monovalent cations exhibited inwardrectification because the sum of endogenous K⁺ and Na⁺ concentrations ishigh in Xenopus oocytes. In addition, Ca²⁺-evoked currents were slightlylower in the presence of 100 mM Na⁺ than in its absence (FIG. 7B),presumably due to the presence of modest competition between Ca²⁺ andNa⁺ for permeation via CaT1. With prolonged application of Ca²⁺ (30minutes) to non-clamped oocytes expressing CaT1, Ca²⁺ entry was enhancedby extracellular Na⁺ (FIG. 5C).

[0054] In order to determine whether Ca²⁺ entry via CaT1 is associatedwith influx or efflux of other ions, the charge-to-⁴⁵Ca²⁺ influx ratiowas determined in voltage clamped oocytes pre-injected with EGTA (FIG.8A). In the absence of external Na⁺, the calculated ratio was notsignificantly different (FIG. 8B), indicating that permeation of Ca²⁺alone accounts for the observed inward currents. The findings that EGTAinjection increases CaT1 activity and that the calcium-evoked currentdecays upon prolonged calcium application (FIG. 8A) suggest that CaT1 iscontrolled by a feedback regulatory mechanism, possibly throughinteraction of intracellular calcium with the transporter.

[0055] CaT1 is relatively specific for Ca²⁺, showing only moderateability to transport other ions. Despite their weak inhibitorypotencies, Ba²⁺ and Sr²⁺, but not Mg²⁺, evoked CaT1-specific currentsalbeit with much smaller amplitudes (FIG. 9). In EGTA-injected oocytesexpressing CaT1 that were clamped at −50 mV, currents due to addition of5 mM Ba²⁺ and Sr²⁺ represented 12±2% and 20±4% (n=17), respectively, ofthe current evoked by 5 mM Ca²⁺. No significant Sr²⁺-evoked orBa²⁺-evoked currents were observed in control oocytes under similarconditions. Other divalent metal ions, including Fe²⁺, Mn²⁺, Zn²⁺, Co²⁺,Ni²⁺, Cu²⁺, Pb²⁺ and Cd²⁺, and the trivalent metal ions Fe³⁺, La³⁺ andGd³⁺ (each at 100 μM), did not evoke measurable currents when applied tooocytes expressing CaT1. In agreement with their inhibitory effects on⁴⁵Ca²⁺ uptake (see FIG. 4E), Gd³⁺, La³⁺, Cu²⁺, Pb²⁺, Cd²⁺, Co²⁺ and Ni²⁺(each at 100 μM) all inhibited the Ca²⁺-evoked currents, whereas thesame concentration of Fe³⁺, Mn²⁺ and Zn²⁺ had no observable effects.Magnesium is neither a substrate (up to 20 mM) nor an effective blockerof CaT1.

[0056] Patch-clamp methodology was employed to search for single-channelactivities using cell-attached and excised membrane patches. Patchpipettes were prepared from 7052 Corning glass capillaries. The pipettetip resistance was 5-10 MΩ. Seal resistances of >10 GΩ were employed insingle channel experiments, and currents were measured using anintegrating patch-clamp amplifier with filtering at 3 kHz through an8-pole Bessel filter. In cell-attached patches, the resting potentialcorresponded to holding the patches at 0 mV. For data acquisition andanalysis, voltage stimuli were applied and single channel currentsdigitized (50-200 us per point) and analyzed using a PC, a Digidata Packand programs based on pCLAMP 6.

[0057] No CaT1-specific channel activities could be identified that wereclearly distinguishable from the endogenous channels present in controloocytes, based on studies of 52 patches from 46 oocytes (EGTA- ornon-EGTA-injected) obtained from seven frogs.

[0058] 6 Applications of CaT1 and its Nucleic Acids

[0059] Although the full potential of CaT1 as a therapeutic target hasnot been investigated, the tissue distribution described above indicatesseveral worthwhile treatment applications involving activation orinhibition of the CaT1 protein. Inhibition of CaT1, for example, may beused to treat kidney stones and various hypercalcemia conditions byrestricting intestinal uptake of calcium. Stimulation of intestinalcalcium uptake, on the other hand, could be used to treat conditions(such as osteoporosis and osteomalacia) characterized by reducedintestinal calcium absorption or reduced bone mass, as well as skindiseases (by stimulation of differentiation) and, possibly, hair growth.Given the potential role of calcium transport in various malignancies,modulation of CaT1 may prove useful in combating tumors.

[0060] CaT1 may be inhibited by pharmacological antagonists, blockingantibodies or by reducing transcription of its gene. Conversely, CaT1may be stimulated by pharmacological agonists, stimulatory antibodies orby increasing transcription of its gene. Blocking or stimulatoryantibodies against CaT1 are obtained in accordance with well-knownimmunological techniques, and a polyclonal mixture of such antibodies isscreened for clones that exert an inhibitory or stimulatory effect onCaT1. The effect of pharmacological compounds or antibodies can bemeasured (and the efficacy of the treatment agent assayed) by observingthe free cystolic calcium concentration (e.g., using a calcium-sensitiveintracellular dye), activation of any calcium-sensitive intracellularprocesses (e.g., the activities of enzymes, gene expression, ionchannels, the activity of other calcium-regulated transporters, orelectrophysiological measurements (as described above)), or ⁴⁵Ca-uptakestudies (also as described above). The monoclonal antibody lines arethen employed therapeutically in accordance with known inhibitorytreatment methodologies. Alternatively, nucleic acid isolated orsynthesized for complementarity to the sequences described herein can beused as anti-sense genes to prevent the expression of CaT1. For example,complementary DNA may be loaded into a suitable carrier such as aliposome for introduction into a cell. A nucleic acid having 8 or morenucleotides is capable of binding to genomic nucleic acid or mRNA.Preferably, the anti-sense nucleic acid comprises 30 or more nucleotidesto provide necessary stability to a hybridization product with genomicDNA or mRNA.

[0061] Nucleic acid synthesized in accordance with the sequencesdescribed herein also have utility to generate CaT1 polypeptides orportions thereof. Nucleic acid exemplified by Seq. I.D. Nos. 1 or 3 canbe cloned in suitable vectors or used to isolate nucleic acid. Theisolated nucleic acid is combined with suitable DNA linkers andpromoters, and cloned in a suitable vector. The vector can be used totransform a host organism such as E. Coli and to express the encodedpolypeptide for isolation.

[0062] Although the present invention has been described with referenceto specific details, it is not intended that such details should beregarded as limitations upon the scope of the invention, except as andto the extent that they are included in the accompanying claims.

What is claimed is:
 18. A method of inhibiting uptake of calcium bycells, the method comprising the steps of: a. providing a first nucleicacid that is complementary to at least a portion of a second nucleicacid encoding a protein that transports calcium across a membrane, thesecond nucleic acid comprising a nucleotide sequence corresponding toSEQ ID NO:1, or substitutions or modifications of the sequence, whereinthe substituted or modified protein transports calcium across a cellularmembrane and is at least 75% identical to SEQ ID NO:2 but distinct fromEcaC; and b. introducing the first nucleic acid into the cells, wherebythe first nucleic acid blocks functional expression of a polypeptideencoded by the second nucleic acid.